The long-term objective is to gain insight into the genetic basis of organismal differences. To achieve such insight it is essential to compare the genes of individuals, populations, and species that differ in known ways and to known extents at the organismal level. The specific aims are to examine (a) three specific cases of regulatory evolution and (b) the relation between evolution at the gene level and evolution at higher levels of biological organization in a broad range of vertebrates. The three cases involve species differing in lysozyme concentration, whose relevance to adaptive evolution at the organismal level is evident or testable. In each case, there is a group of species that has an approximately 100-fold elevation of the lysozyme level in a specific part of the digestive tract, and a closely related group with a low level. The lysozyme genes and their regulatory elements will be compared in the two groups by cloning and structural analysis, as well as by functional tests with the cloned DNA, to find out what types of genetic change are responsible for the major differences in the pattern of lysozyme expression. Three measures of divergence at the gene level will be investigated: (1) degree of regulatory difference as estimated by two-dimensional gel electrophoresis of hundreds of proteins detectable in extracts of specific tissues, (2) number of rounds of concerted evolution in rRNA gene clusters, and (3) extent of point-mutational divergence in proteins. (There is considerable published evidence on the third measure of genetic divergence.) Three measures of divergence at higher levels of organization will also be considered: (1) overall degree of morphological difference, (2) degree of karyotypic difference, and (3) number of speciation events. Divergence at these higher levels will be estimated from this laboratory's morphological measurements and from surveys of karyotypic, taxonomic, and fossil literature. Attention will center on the comparison of rates of divergence in these six respects for numerous species of mammals and amphibians. The main question to be addressed is whether the high rates of organismal evolution that are characteristic of mammals (particularly the higher primates) have been paralleled by high rates of concerted evolution or overall change in pattern of gene expression. This research is therefore expected to contribute to knowledge of the types of mutation that account for the organismal differences among individuals, populations, and species.